Giorgio Sartor

Characterization of cholinesterase activity in three bivalves inhabiting the North Adriatic sea and their possible use as sentinel organisms for biosurveillance programmes

Anticholinesterases constitute a major portion of modern synthetic insecticides and the assessment of cholinesterase (ChE) inhibition is widely used as a specific biomarker for evaluating the exposure of non-target organisms to these pollutants. To evaluate the possible exposure of the North Adriatic sea coastal environment to residues of agriculture practices, we ascertained whether the oyster, Ostrea edulis and the clam, Tapes philippinarum, highly valuable resources commercially harvested in the area, could be selected as sentinel species. We characterized the ChE biochemical properties in their gills, and for comparison the analysis was carried out also in gills of the mussel, Mytilus galloprovincialis, extensively used as a biological indicator of water quality. In the oysters and mussels, the ChE activity was a function of increasing concentrations of substrate in the range 0.01-0.5 mM. K(m) values were 93+/-15 and 77+/-8 microM for O. edulis and M. galloprovincialis, respectively. No detectable ChE activity was found in gills of T. philippinarum, at any tested condition. Significant inhibition of ChE activity by eserine, carbaryl, and ethyl-paraoxon was observed in gills of O. edulis and M. galloprovincialis. Although field validation is needed, the present study suggests that O. edulis may be employed as a biological indicator for assessing pesticide contamination, whilst T. philippinarum does not seem useful for this purpose.

Introduction of oxygenated side chain into imidazolium ionic liquids: Evaluation of the effects at different biological organization levels

The biological effects of a class of oxygenated imidazolium ionic liquids were studied in comparison with alkyl imidazolium salts (BMIM BF4 and BMIM N(CN)2).The cellular and subcellular effects were evaluated on rat pheochromocytoma PC12 cell lines, through MTT test, lactate dehydrogenase release and acetylcholinesterase inhibition; the eco-toxicological responses were assessed through the acute toxicity tests towards Daphnia magna and Vibrio fischeri. The introduction of ethoxy moieties in the lateral chain of imidazolium cations reduced the biological effects in all the tests. The acute toxicity towards D. magna was not affected by the number of ethoxy units, but the crustacean seemed to be sensitive to the type of anion; on the contrary, a further addition of ethoxy moieties increased the toxicity towards V. fischeri, M(OE)4MIM N(CN)2 being the most toxic oxygenated ionic liquid. In the cytotoxicity assays the salts with oxygenated cations resulted ineffective compared to BMIMs, independently from the anion and the number of ethoxy units in the lateral chain. In order to estimate the influence on membrane fluidity, an analysis of fluorescence anisotropy was done and it indicated that BMIM BF4, the most toxic ionic liquid among the tested ones, led to a destabilization of the model membranes at any molarity.

Cd2+ and Hg2+ affect glucose release and cAMP-dependent transduction pathway in isolated eel hepatocytes.

Isolated hepatocytes of the European eel (Anguilla anguilla) have been used as experimental model to characterize the effects of Cd(2+) and Hg(2+) on either basal or epinephrine-stimulated glucose release. Cd(2+) strongly reduced glucose output from cells perifused in BioGel P4 columns and challenged with epinephrine, with a maximum inhibition of 95% reached at 10 microM (IC(50) 0.04 microM). The epinephrine-stimulated glucose output was also reduced by Hg(2+), although a significant inhibition of about 60% was achieved only at 10 microM (IC(50) 5 microM). The possible influence of Cd(2+) and Hg(2+) on adenylyl cyclase/cAMP transduction pathway has been investigated, since this system is known to play a pivotal role in the regulation of fish liver glycogen breakdown and consequent glucose release. Micromolar concentrations of both heavy metals significantly reduced the epinephrine-modulated cAMP levels in isolated eel hepatocytes, in good agreement with the reduction of glucose output. Cd(2+) and Hg(2+) also significantly reduced basal and epinephrine-stimulated adenylyl cyclase activity in liver membrane preparations. A competitive inhibition with respect to Mg(2+) was shown by Cd(2+) and Hg(2+), which significantly reduced the affinity of the allosteric activator for the adenylyl cyclase system. Apparent Km for Mg(2+) was 4.35 mM in basal conditions, and increased to 9.1 and 7.1 mM in the presence of 10 microM Cd(2+) and Hg(2+), respectively. These results indicate that Cd(2+) and Hg(2+) may impair a crucial intracellular transduction pathway involved in the adrenergic control of glucose metabolism, but also in several other routes of hormonal regulation of liver functions.

Lipid Peroxidation in Cancer Cells: Chemical and Physical Studies

Our studies on the biochemical composition and the structural organization of smooth and rough endoplasmic reticulum isolated from Morris hepatomas 9618A and 3924A confirm the results obtained employing the total microsomal fraction. We have definitely established the following facts: (1) Tumor subcellular organelles exhibit the very low degree of peroxidizability that has been shown to be related to the growth rate of the tumor. (2) Associated with such a low susceptibility to peroxidation are (a) changed lipid composition of cellular membranes, whose content in polyunsaturated fatty acid is markedly decreased, and (b) changed static and dynamic properties of the membrane. Previously it was also found that cellular oxy-radical scavenging enzymes are markedly reduced. From these data, it is possible to infer that tumor membranes are altered structurally and functionally in part as the result of an oxy-radical-induced damage that occurs in vivo under conditions of oxygen toxicity. This seems to be supported by recent findings that the spontaneous increase in growth rate of the originally very slow-growing Morris hepatoma 9618A results also in the loss of cytochrome P-450 (an important intramembraneous propagator of lipid peroxidation) as well as of C20:4 and C22:6. Studies performed by GLC and GC-MS on the fatty acid residues of phospholipids of rat liver microsomes show the presence of C20:3-OH and C18:1-OH, but no hydroxyl derivatives of low molecular weight aldehydes. The hydroxyl derivatives of arachidonic acid and linoleic acid are present in much smaller amounts in the microsomes isolated from H9618A and H3924A.

On the interactions of metal ions with tryptophan and glyciltryptophan: a fluorescence study

Abstract The interaction of copper (Cu2+) and nickel (Ni2+) with tryptophan (Trp) and glyciltryptophan (GlyTrp) was studied by monitoring the quenching of fluorescence due to complex formation. pH titration of mixtures of M2+/L show that Cu2+ lowers the pKa for the NH+3 group from 9.36 to 5.9 in Trp and from 8.2 to 5.5 in Glytrp while the effect of Ni2+ is relatively smaller. Association constants were evaluated from Stern-Volmer plots and are shown to be 1.9×105 M-5 for Cu2+-Trp, 4.4×104 M-1 for Cu2+-GlyTrp, 4.0×104 M-1 for Ni2+-Trp and 6.9×103 M-1 for Ni2+-GlyTrp measured at pH 9. The binding of metals to Trp or GlyTrp at pH 7 leads to very different constants for Ni2+ and Cu2+, due to enhanced complex formation between the aniomic form of the compounds and the metal ions. The binding of nickel at this pH is strongly reduced.

Tryptophan interactions of gramicidin A′ channels in lipids: a time-resolved fluorescence study

The evolution of the incorporation of cation transport channels into lysolecithin micelles by gramicidin A was followed by measuring the ns time-resolved fluorescence of the tryptophan residues. In all samples, the tryptophan fluorescence could be resolved into three exponentially decaying components. The three decay times ranged from 6 to 8 ns, 1.8 to 3 ns, and 0.3 to 0.8 ns, depending on the emission wavelength. The fractional fluorescence of each component changed with incubation time. The long lifetime component had a reduced contribution to the total fluorescence while the short decay time component increased. The fluorescence spectra could be resolved into three distinct fluorescent components having maxima at 340 nm, 330 nm and 323 nm after 90 min of incubation, and 335 nm, 325 nm and 320 nm after 24 h of incubation. These maxima were, respectively, associated with the long, medium and short decay components. The fluorescence decay behaviour was interpreted as representing three families of tryptophans, the short lifetime component being due to a stacking interaction between tryptophan residues. The variation with incubation time suggests a two-step process in the channel-lipid organization. The first is associated with the conformational change of the polypeptide as it takes up a left-handed helical head-to-head dimer structure in the lipid. The second step is proposed to involve changes originating from membrane assembly and intermolecular interactions between channels as they form hexameric clusters.

Interaction of copper and nickel ions with tryptophan and glyciltryptophan: Time resolved flourescence study

Abstract The interaction of copper (Cu++) and nickel (Ni++) with tryptophan (Trp) and glyciltryptophan (GlyTrp) was studied monitoring the quenching of flourescence due to complex formation. The deviations from linear Stern-Volmer plots for both metals were treated in a different way reflecting the difference in binding. In the case of nickel the Lehrer formalism was used which permitted us to estimate the binding constant as well as the fraction of fluorophore accessible to the metal. In the case of copper the Hill plot formalism was more appropriate giving a degree of cooperativity upon binding as well as the association constant of the complex. The values of the constants are in agreement with our previous values based on Stern-Volmer plots. Time resolved fluorescence measurement were also performed to assess the mechanism of quenching as well as the contribution from different conformers in solution. The fluoresscence decay is best described as a triple exponential with lifetimes of 7.9, 2.7 and 0.23 ns at 25°C. The quenching is essentially static, the lifetimes remain constant and changes are observed only in the pre-exponentials. In the case of nickel the shortest lifetime remains essentially unquenched while the other two components are effectively quenched. In the case of copper the quenching depends on the metal concentration but all of the conformers are quenched significantly. Our results suggest the assignment of one of the conformers as being the short lifetime component in the decay of fluorescence.

Effect of CoQ Homologues on the Fluidity of Phospholipid Bilayers as Studied by Fluorescence Polarization of Perylene

The effect of Coenzyme Q analogues on the fluidity of egg lecithin bilayers was studied by fluorescence polarization of perylene. The parameter was clearly affected by the side-chain length of the quinones, by their concentration, and by their redox state. With increasing concentration, short-chain quinones decreased the bilayer fluidity, and did so to an even greater extent in their reduced state. On the contrary, long-chain analogues fluidized the bilayer, the effect being independent of their redox state. Such observations might explain the lower efficiency of short-chain quinones, as compared to the long-chain analogues, in restoring, in vitro, the respiratory activity of CoQ2-depleted mitochondria.

The Role of the Side Chain in the Antioxidant Activity of Ubiquinones

The question has been addressed whether the side chain contributes to the antioxidant activity of Ubiquinones. The length, the chemical composition and structure of the chain have been considered. The effect of the actual concentration of the quinone in egg lecithin vesicles has been investigated by means of both UV spectroscopy and time resolved fluorescence quenching experiments of 12-AS. The results indicate that the antioxidant properties of the quinone do not seem to depend on the side chain.

Lipid Composition, Physical State, and Lipid Peroxidation of Tumor Membranes

Studies were carried out on microsomes isolated from the highly differentiated (slow-growing) Morris hepatoma 9618A, on microsomes and plasma membranes from the poorly differentiated (fast-growing) Morris hepatoma 3924A, and rat liver used as control. The lipid composition (phospho-lipid and cholesterol content, degree of fatty acid unsaturation) and peroxidation of such membranes has been correlated with the order and fluidity of the membrane bilayer. The results indicate that substrate availability is the rate-limiting step in microsomal and plasma membrane lipid peroxidation of hepatoma 3924A. From diphenylhexatriene fluorescence depolarization measurements it appears that the changes in lipid composition cause an increase in the order of the lipid bilayer on going from the control to hepatoma 9618A and 3924A microsomes, while fluidity is virtually unchanged. Conversely, for similar chemical changes, in plasma membranes from hepatoma 3924A the order is nearly the same and there is a decrease in fluidity. The changes in the above parameters of tumor membranes might be partly related to the loss of protective enzymes against oxygen radicals. This is supported by the observation that inhibition of liver superoxide dismutase and glutathione reductase, by treatment of rats with diethyldithiocarbamate and chloroethyl nitrosourea, respectively, renders the microsomal membranes more resistant to lipid peroxidation in vitro.